基于高灵敏轮廓分析的粗糙脉孢菌ERG4催化甾醇合成路径研究

ERG4 is a key enzyme in ergosterol biosynthesis pathway. In previous study, we found erg4 deletion mutant has slower growth rate than the wild type strain and is hypersensitive to antifungal azoles. This suggests that C-24 reductase encoded by erg4 could be a potential target of antifungal drugs in filamentous fungi. By using the combined method of molecular biology and analytical chemistry, ten new sterol metabolites related to the biosynthesis of ERG4 were found by a high sensitive sterol profiling analysis. Furthermore, the function of erg4 in ergosterol biosynthesis was validated by gene complementation. This project will identify the ERG4-derived metabolites in the erg4, erg5 mutants and the wild type strains based on the high performance liquid chromatography and nuclear magnetic resonance spectrometer. Analyze catalytic activity of ERG4 in sterol biosynthesis in vitro and finally identify the ERG4-derived sterols important for hyphal growth by supplementation with the sterols which have significant difference in erg4 mutant compared with wild type. This study will provide important theoretical basis for the development and the research on the mechanism of the new antifungal drugs.

ERG4是麦角甾醇合成路径中的关键酶，本课题组在前期研究中发现，粗糙脉孢菌erg4基因的缺失突变会导致生长减慢，对唑类药物敏感性增强。这一现象表明erg4所编码的C-24还原酶可以作为丝状真菌抗真菌药物的一个潜在作用靶点。采用分子生物学与分析化学相结合的方法，我们已经利用高灵敏的甾醇轮廓分析方法发现了10个与erg4基因产物密切相关的新麦角甾类代谢物，并在此基础上用基因回补的方法初步验证了突变基因在相关代谢途径中的作用。本研究拟放大发酵erg4、erg5以及野生型菌株，制备、鉴定所关注的麦角甾类代谢物；通过体外反应实验测定ERG4体外活性以最终确定所推测的代谢路径；分别将erg4基因缺失时显著性变化的代谢物加入培养基中，确定影响粗糙脉孢菌正常生长的关键代谢物。本项目一旦实施，将为新的抗真菌药物的开发及其作用机制的研究提供重要的理论基础。

麦角甾醇是真菌细胞膜的主要成分，ERG4是麦角甾醇合成路径中的关键酶。本课题组采用分子生物学与分析化学相结合的方法，利用高灵敏的甾醇轮廓分析方法，发现了与erg4基因产物密切相关的麦角甾类差异代谢产物；并通过优化发酵条件以及色谱制备等方法制备得到了11种麦角甾类差异代谢产物。随后，我们通过高分辨质谱以及核磁共振等技术手段对其进行分析鉴定，进一步完善了麦角甾醇的生物合成路径。对erg4基因代谢路径的研究和完善将为新的抗真菌药物的开发及其作用机制的研究提供重要的理论基础。在此基础上分析和整理数据，申请专利1篇，发表SCI文章1篇。